TRIM44 promotes autophagy through SQSTM1 oligomerization in the response to oxidative stress induced by Arsenic Trioxide in cancer cells

Tripartite motif containing 44 (TRIM44), a deubiquitinase, plays a pivotal role in connecting proteotoxic stress response to autophagic degradation in cancer and neurological diseases. While numerous studies have reported the upregulation of TRIM44 as a prognostic maker in various cancers, the detailed molecular mechanisms through which TRIM44 promotes autophagic degradation remain unclear. Here, we reported that TRIM44 can promote autophagy in response to oxidative stress which results in decreased cytotoxicity in Arsenic Trioxide treated cancer cells. The study focuses on the posttranslational modi�cation of sequestosome-1 (SQSTM1) and its role in enhancing sequestration function during autophagic degradation. We discovered that TRIM44 signi�cantly promotes SQSTM1 oligomerization in PB1 domain-dependent and oxidation-dependent manners. Furthermore, TRIM44 enhances the interaction between protein kinase A (PKA) and oligomerized SQSTM1, leading to increased phosphorylation of SQSTM1 at S349 and subsequent activation of NFE2L2 in response to oxidative stress. Collectively, our data support the potential roles of TRIM44 in the sensitivity of SQSTM1-mediated autophagy in the context of cancer, ageing and ageing-associated diseases, as well as neurodegenerative diseases.


Introduction
Pathological processes such as cancer, ageing, and ageing-related diseases, and neurodegenerative diseases are commonly associated oxidative damage to DNA and proteins [1][2][3][4][5].Cellular homeostasis is maintained through a myriad of mechanisms, including autophagy, which functions to degrade damaged proteins and organelles that could be potentially toxic under basal conditions [6].Furthermore, autophagy is pivotal in ensuring cell survival under various stress conditions including chemotherapy-induced oxidative stress [7].Since arsenic trioxide (As[III]) was rst approved as the front-line therapy for acute promyelocytic leukemia, its anti-cancer properties for various malignancies have been under intense investigation [8].As [III] has been proven to be effective not only in the treatment of chronic lymphocytic leukemia and acute myeloid leukemia, but also for some solid tumors, such as cervical cancer, hepatocellular carcinoma, head and neck cancers, lung cancer, stomach cancer, and so on [9].Autophagy is activated in response to oxidative stress induced by As [III] to protect the cells from apoptosis [10].
The KEAP1-NFE2L2 system is a well-established cellular defense mechanism against oxidative stress and electrophilic disturbances [11].Modi cations of KEAP1 cysteine residues result in its degradation, which stabilizes NFE2L2, leading to its translocation to the nucleus and subsequent transcriptional activation of cytoprotective genes through its heterodimerization with small Maf proteins.Beyond this canonical pathway, SQSTM1, a recognized autophagic adaptor, binds to the NFE2L2-binding site on KEAP1, competitively inhibiting the KEAP1-NFE2L2 interaction, critical for the transcriptional of genes encoding antioxidant proteins and anti-in ammatory enzymes [12,13].However, the pathogenic implications of KEAP1-NFE2L2 regulation by SQSTM1 and its connection with autophagic degradation are not fully clari ed.
TRIM family members are involved in a spectrum of cellular functions, including regulation of the immune system, antiviral responses, autophagy-related receptor regulation, and cancer initiation [14].
Distinguishing itself within the TRIM family proteins, TRIM44 possesses a zinc nger ubiquitin protease domain (ZF-UBP) at its N-terminus, functioning as a deubiquitinating enzyme, as opposed to the more common RING domain [15].Since its initial discovery in 2001 [16], TRIM44 has been found to be upregulated in multiple human tumors [17][18][19][20] and is associated with a poor prognosis.However, the detailed molecular mechanisms of TRIM44's role in tumor initiation and/or progression remain elusive.
Our previous research indicated that TRIM44 plays an important role in promoting autophagy by facilitating the oligomerization of SQSTM1 2001 [21].Therapeutically targeting autophagy is showing promise in cancer patients [22].However, the precise mechanism by which TRIM44 augments SQSTM1 oligomerization needs further elucidation.
In this study, we demonstrate that TRIM44 promotes the oligomerization of SQSTM1, dependent on its deubiquitinating activity.Moreover, the oligomerization of SQSTM1 mediated by TRIM44, is PB1 domaindependent and oxidation-dependent.This TRIM44-driven oligomerization fosters the phosphorylation of SQSTM1 at S349 via PKA signaling, leading to the sequestration of KEAP1 and subsequent activation of the NFE2L2-mediated antioxidant response.Thus, TRIM44 plays a substantial role in SQSTM1-mediated autophagy and protecting cells from oxidative stresses by enhancing the autophagic process.

TRIM44 alleviates cytotoxicity via enhancing autophagy in response to oxidative stress
To investigate whether TRIM44 plays a key role in promoting cancer cell survival under oxidative stress induced by Arsenic trioxide, we engineered cell lines with either upregulated (TRIM44[OE]) or downregulated TRIM44 (TRIM44[KD]) using a lentiviral system as full knockout proved lethal [23] (Supplementary Fig. S1A) and treated cells with Arsenic trioxide.Cells overexpressing TRIM44 exhibited markedly increased resistance to As[III]-induced cytotoxicity compared to control cells.In contrast, TRIM44 knockdown resulted in reduced resistance to As[III] (Fig. 1A-C), underscoring TRIM44's potential role in cell survival upon exposure to oxidative stress.
Given that TRIM44 has recently been identi ed as a novel link connecting the UPS system with the autophagy degradation pathway [21] and it has been shown that autophagy is activated in response to oxidative stress to protect the cells from apoptosis, we assessed autophagy activity in TRIM44 [OE] and TRIM44[KD] cancer cells under oxidative stress.Following As[III] treatment, we observed a signi cant increase in LC3-II levels in TRIM44[OE] cells compared to control cells.However, these levels markedly decreased in TRIM44[KD] cells (Fig. 1D), showing TRIM44's important role in modulating the autophagy pathway following oxidative stress.
We also explored the impact of TRIM44-mediated autophagy in mitigating oxidative stress.The addition of 3-MA, an autophagy inhibitor, along with As[III] resulted in a notable increase in apoptosis.This effect was signi cantly distinct from that observed in individual treatments, indicating the critical role of TRIM44-induced autophagy in reducing apoptosis rates under oxidative stress (Fig. 1E).Collectively, our data supported that TRIM44 attenuates cytotoxicity via enhancing autophagy in response to oxidative stress.

TRIM44 promotes the oligomerization of SQSTM1
TRIM44 is a three-domain protein consisting of an N-terminal zinc-nger UBP (ZF) with deubiquitinating activity, a b-box (BB) domain, and a coiled-coil domain (CC) (Fig. 2A), diverges from the typical E3 ligase function attributed to TRIM family members [24,25].Our prior research pinpointed a critical role for TRIM44 in augmenting autophagy by facilitating SQSTM1 oligomerization [21].Co-transfection experiments with HA-tagged SQSTM1 and the full-length (FL) form of TRIM44, followed by immuno uorescence analysis, revealed a pronounced increase in SQSTM1 oligomerization, compared to the GFP control (Fig. 2B and C).Conversely, cells expressing a truncated TRIM44 lacking the ZF domain (TRIM44ΔZF) displayed a marked reduction in SQSTM1 oligomerization, underscoring the essential role of the ZF domain's deubiquitinating activity in mediating this process (Fig. 2B and C).
Under non-reducing conditions, monomeric SQSTM1 typically presents as a 62 kDa band, which shifts to higher molecular weight complexes upon oligomerization or protein interaction [26].To further delineate TRIM44's effect on SQSTM1 oligomerization, we analyzed the SQSTM1 oligomerization in TRIM44[OE] cells.Overexpression of TRIM44 was found to enhance SQSTM1 oligomerization, whereas its knockdown reduced oligomerization following oxidative stress (Supplementary Fig. S1B).The ubiquitination of SQSTM1 hinders its dimerization and subsequent formation of oligomerization [26].To explore this, we conducted a deubiquitylation assay using exogenously expressed SQSTM1 and TRIM44.We observed that TRIM44 expression decreased the level of ubiquitinated SQSTM1, while the inhibition of TRIM44 resulted in increased SQSTM1 ubiquitination (Supplementary Fig. S1C).Collectively, these ndings support that TRIM44 deubiquitinating activity facilitates the oligomerization of SQSTM1.
To further elucidate the mechanism by which TRIM44 promotes the oligomerization of SQSTM1, we introduced mutations that disrupt oligomerization (D69A, K7R, or double mutation K7AD69A), which impeded dimerization and subsequent oligomer formation [26,27].Remarkably, even with these mutations, oligomerization was signi cantly elevated in the presence of TRIM44, contrasting with GFP controls (Fig. 2D and E).Correspondingly, the reduced levels of ubiquitination from these mutations were con rmed (Fig. 2F, Supplementary Fig. S1D-G), aligning with previous studies [26 -27].We also introduced the Phox and Bem1p domain (PB1) -deleted form (ΔPB1) of SQSTM1, which is known to be required for self-oligomerization through interactions with other SQSTM1 molecules [28].While deletion of the PB1 domain drastically reduced SQSTM1 oligomerization, TRIM44 still signi cantly boosted the oligomerization of this truncated form (Fig. 2G to I).Taken together, these data indicate that TRIM44 augments the oligomerization of SQSTM1 not only through the PB1 domain, highlighting TRIM44's signi cant in uence on the oligomerization and subsequent functional modulation of SQSTM1.
TRIM44 enhances SQSTM1 oligomerization in response to oxidative stresses Oligomerization of SQSTM1 is facilitated by disulphide-linked conjugates (DLC) formation under oxidative stress conditions [29].To investigate the alternative mechanism(s) of TRIM44-mediated oligomerization of SQSTM1, we exposed cultured cells to a range of treatments and analyzed them by immuno uorescence staining (Fig. 3A).We observed an increase in intracellular SQSTM1 aggregates in cells overexpressing TRIM44 (TRIM44[OE]) compared to control cells (TRIM44[OE-CON]) without any treatment (Fig. 3A).The presence of autophagy inhibitors like Ba lomycin A1 (Baf A1), which blocks lysosomal acidi cation and degradation without affecting autophagosome-lysosome fusion [30], or Chloroquine (CQ), which affecting autophagosome-lysosome fusion, led to an intensi ed accumulation of SQSTM1 aggregates in TRIM44[OE] cells (Fig. 3A).This phenomenon was similarly observed under oxidative stress conditions induced by hydrogen peroxide (H 2 O 2 ) or arsenic trioxide (As[III]), with TRIM44[OE] cells displaying a higher accumulation of SQSTM1 aggregates compared to TRIM44[OE-CON] cells (Fig. 3A).Conversely, the knockdown of TRIM44 resulted in a reduced formation of SQSTM1 aggregates under these treatments (Fig. 3A), implying that TRIM44 may be involved in the oligomerization-promoting DLC formation in SQSTM1 under oxidative stresses.
To further probe TRIM44's impact on the oligomerization of SQSTM1 primed by DLC formation, a DLCformation de cient SQSTM1 mutation (C105/C113A) was employed in our immuno uorescence staining.The levels of oligomerization from this mutation were both markedly reduced in GFP-positive cells and GFP-TRIM44-positive cells, suggesting that DLC formation promotes SQSTM1 oligomerization and its a nity for ubiquitin-positive aggregates under oxidative stress, and the oligomerization of SQSTM1 mediated by TRIM44 is partly derived from DLC formation (Fig. 3B).The application of the reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC) resulted in a decrease in SQSTM1 oligomerization levels, con rming the response of SQSTM1 oligomerization to oxidative stress (Fig. 3C-E).Moreover, GFP-TRIM44-positive cells showed a more pronounced reduction in SQSTM1 oligomerization when treated with NAC, indicating that TRIM44 enhances SQSTM1 oligomerization not solely via deubiquitinating activity but also by increasing intracellular ROS levels (Fig. 3C-E).Taken together, these ndings point to TRIM44's capacity to modulate SQSTM1 oligomerization through both the PB1 domain and oxidation of SQSTM1.

Oligomerization of SQSTM1 is essential for its phosphorylation
The capacity of SQSTM1 to adjust to redox shifts underpins its aggregation, particularly under oxidative stress -a mechanism crucial for vertebrate survival, providing defense during ageing [29].Building on previous studies that identi ed phosphorylation sites on SQSTM1 at S349, S403, and S407, which are critical for substrate binding [31,32,33], we sought to explore the interplay between redox sensing-driven SQSTM1 oligomerization and its phosphorylation patterns.To this end, HeLa cells stably expressing either wild-type SQSTM1 or its oxidation-insensitive mutations (C105/113A, K102E) were treated with As[III] and analyzed by western blotting under non-reducing and reducing conditions.These two mutations exhibited a marked reduction in oligomerization, displaying a dose-dependent decline posttreatment, thereby underscoring the redox-sensitivity of SQSTM1 as a determinant of its oligomerization during oxidative stress (Fig. 4A).
Oligomerization of SQSTM1 precedes its sequential phosphorylation at serine residues (S407, S403), followed by phosphorylation at S349 within its UBA domain.The phosphorylation at S349 enhances SQSTM1's binding to KEAP1, leading to the release of NFE2L2 from KEAP1 and subsequent activation of NFE2L2's targets involved in antioxidant and anti-in ammation processes [31,34].Our data showed that hindered SQSTM1 oligomerization led to attenuated phosphorylation at S349 (Fig. 4B and C).On the other side, the phosphorylation-de cient mutation SQSTM1 S349A did not prevent SQSTM1 oligomerization (Fig. 4D and E), supporting that SQSTM1 oligomerization is a prerequisite for the phosphorylation of SQSTM1 S349.

TRIM44 enhances phosphorylation of SQSTM1 at S349 via PKA
Given that TRIM44 promotes SQSTM1 oligomerization, and SQSTM1 oligomerization is required for its phosphorylation, we delved into the mechanism by which TRIM44 regulates the phosphorylation of SQSTM1 at S349.Enhanced phosphorylation at this site was observed in cells with TRIM44 overexpression (TRIM44[OE]), while a reduction was noted upon TRIM44 knockdown (TRIM44[KD]) (Supplementary Fig. S2A and B).When challenged with As[III], TRIM44[OE] cells exhibited a pronounced increase in SQSTM1 S349 phosphorylation compared to the control group (TRIM44[OE-CON]) (Fig. 4C).
The kinase responsible for phosphorylating SQSTM1 at S349 has been a subject of debate.Although the involvement of the mammalian target of rapamycin (mTOR) in the phosphorylation at this site has been reported [31], our data indicated that suppression of mTOR activity by an inhibitor pp242 (mTORi), did not abolish TRIM44's enhancement of SQSTM1 phosphorylation under either basal or oxidative conditions (Supplementary Fig S2D and F).This holds true even though TRIM44[OE] cells exhibited suppressed mTOR activity (Supplementary Fig. S2C).
A previous study identi ed initial phosphorylation of serine 407 in SQSTM1's UBA domain by the Unc-51like autophagy activating kinase 1 (ULK1).Subsequent phosphorylation at serine 403 by either casein kinase 2 (CK2), TANK-binding kinase 1 (TBK1), or ULK1 enhances SQSTM1's binding a nity for ubiquitinchains [34].On this basis, we investigated whether TRIM44 in uences SQSTM1's pS349 via these kinases.Our results con rmed that inhibiting these kinases did not alter the TRIM44-mediated increase in SQSTM1 pS349 levels in TRIM44[OE] cells (Supplementary Fig. S2D).Also, we did not see any increased level of these kinases in TRIM44[OE] cells and decreased levels of these kinases in TRIM44[KD] cells (Supplementary Fig. S2E).
To further investigate into the mechanism behind TRIM44-mediated phosphorylation of SQSTM1 at S349, NetPhos was initially employed to predict the potential kinase targeting SQSTM1 S349.
Additionally, kinases interacting with TRIM44 were screened from our mass spectrometry data (unpublished).From these ndings, we focused on four candidates glycogen synthase kinase 3 (GSK3), Ca 2+ /calmodulin-dependent protein kinase II (CaM-II), protein kinase A (PKA) and casein kinase 1 (CKI).Screening with inhibitors speci c to these kinases in TRIM44[OE] cells revealed that only inhibition of PKA suppressed the TRIM44-induced phosphorylation at S349, while inhibitors of GSK3, CaM-II, and CKI had no signi cant effect (Fig. 5A and Supplementary Fig. S2G).This was corroborated by a signi cant increase in PKA activity in TRIM44[OE] cells and a decrease in TRIM44[KD] cells (Fig. 5B), with a notable increase in SQSTM1 pS349 in the anti-phospho-PKA substrate fraction, particularly under oxidative stress (Fig. 5C).
Next, we examined whether PKA catalytic subunit α (PKA Cα) expression might be altered by TRIM44.As shown in Fig. 5D, PKA Cα was reduced in TRIM44[KD] cells under basal and oxidative stress conditions.We further validated the involvement of PKA in TRIM44-mediated SQSTM1 S349 phosphorylation using siRNA transfection.Silencing PKA Cα resulted in a marked reduction in this phosphorylation in TRIM44[OE] cells (Fig. 5E).Conversely, overexpressing PKA Cα in TRIM44[KD] cells restored phosphorylation levels (Supplementary Fig. S2H).Consistent with this, we observed that the interaction between PKA Cα and SQSTM1 was enhanced in TRIM44[OE] cells and a further increase following Ba lomycin A1 (Baf A1) treatment (Fig. 5F).In addition, the a nity of PKA Cα and SQSTM1 was abolished by oligomerization-de cient mutation K7R, supporting that TRIM44 promotes SQSTM1 S349 phosphorylation by enhancing the interaction between PKA and oligomerized SQSTM1 (Supplementary Fig. S2I).These ndings collectively highlight that TRIM44-mediated SQSTM1 phosphorylation at S349 is achieved by upregulating PKA Cα -induced PKA signaling.
TRIM44-induced SQSTM1 oligomerization promotes KEAP1 sequestration and potentiates NFE2L2 activation KEAP1 is a substrate for autophagy, and the interaction of KEAP1 with SQSTM1 is indispensable for its autophagic degradation [35].Phosphorylation of SQSTM1 at serine 349 enhances the interaction between SQSTM1 with KEAP1 [36].This observation introduces the concept that TRIM44 could augment the binding between SQSTM1 and KEAP1 and subsequent degradation of KEAP1.To address this, we initially evaluated TRIM44's in uence on KEAP1 protein levels.Our data revealed that TRIM44 overexpression led to a decline in KEAP1 protein abundance, while suppression of TRIM44 resulted in an increase in KEAP1 (Fig. 6A and supplementary Fig. S3A).
The KEAP1-NFE2L2 pathway is pivotal in the cellular defense against oxidative stress.NFE2L2, as a transcription factor, orchestrates the cellular adaptation to oxidative and electrophilic stress by activating antioxidant genes in response to redox imbalances [37].Under stress conditions, KEAP1 is degraded and leads to the stabilization of NFE2L2, thereby allowing its accumulation and nuclear translocation to initiate the antioxidant transcriptional program [38].Based on our ndings that TRIM44 mediates KEAP1 degradation, we hypothesized that TRIM44 enhances NFE2L2 activity.Consistent with this hypothesis, TRIM44 overexpression correlated with elevated nuclear NFE2L2 level, while TRIM44 silencing reduced these levels, especially following As[III] exposure.This modulation in NFE2L2 localization was inversely related to cytoplasmic KEAP1 levels (Fig. 7A).Furthermore, a concomitant increase in the expression of NFE2L2 target genes, such as the heme oxygenase-1 (HMOX1) gene and thioredoxin (TXN), was observed both with or without As[III] treatment (Fig. 7B).Interestingly, the upregulation of NFE2L2 target genes occurred independently of changes in NFE2L2 mRNA expression, with a pronounced response to As[III] exposure (Fig. 7B, Supplementary Fig. S3G).However, this response was attenuated in TRIM44 knock-down cells (Fig. 7B).
To further validate TRIM44's impact on the KEAP1-NFE2L2 axis, immunoprecipitation assays were performed to examine the interaction between KEAP1 and SQSTM1, as well as the interaction between KEAP1 and NFE2L2 in 293T cells.Our results revealed that an enhanced interaction between KEAP1 and SQSTM1 was observed in TRIM44[OE] cells, compared to control (TRIM44[OE-CON) (Fig. 7C), while the binding between KEAP1 and NFE2L2 was reduced in TRIM44[KD] cells (Fig. 7C).Aligning with these phenomena, TRIM44 disrupted the interaction between NFE2L2 and SQSTM1 (Fig. 7D).
In vertebrates, SQSTM1's redox sensitivity plays a pivotal role in modulating autophagic degradation and cell survival under oxidative stresses [29].Next we explored the effect of TRIM44 on the KEAP1-NFE2L2 pathway in the context of SQSTM1 redox-sensitivity-de cient mutation (C105/113A).In the context of wild-type SQSTM1 (WT), following oxidative stress, nuclear NFE2L2 was signi cantly increased in TRIM44[OE] cells, although considerable nuclear NFE2L2 was detected in TRIM44[OE-CON] cells.On the other hand, in the context of mutation SQSTM1 (C105/113A), following oxidative stress, almost no nuclear NFE2L2 was detected in TRIM44[OE-CON] cells.However, an increase of NFE2L2 level in nuclear fraction was observed in TRIM44[OE] cells (Fig. 7E).
We also investigated the role of PKA-mediated SQSTM1 oligomerization in NFE2L2 activation by TRIM44.The notable increase in NFE2L2 target gene expression mediated by TRIM44 was signi cantly suppressed following PKA Cα knockdown (Supplementary Fig. S4A and B).Conversely, overexpression of PKA Cα rescued the reduction in NFE2L2 target expression caused by TRIM44 silencing (Supplementary Fig. S4C).These ndings support that PKA is involved in the TRIM44-driven cytoprotective activity of NFE2L2.Corroborating our ndings, neither the mTOR inhibitor pp242 nor any other tested inhibitors affected the TRIM44-mediated NFE2L2 target upregulation (Supplementary Fig. S3H).In conclusion, our data support that TRIM44 plays a critical role in facilitating the oligomerization of SQSTM1 through interactions with PKA.This interaction serves as a key mechanism in enhancing the cytoprotective activity of NFE2L2, underscoring the signi cance of TRIM44 in cellular stress response pathways.

Discussion
Oligomerization of SQSTM1 is essential for its targeting to autophagosome formation site and its subsequent sequestration function during autophagic degradation [40][26].So far, two mechanisms have been reported for the formation of SQSTM1 oligomerization.One mechanism is PB1 domain-dependent, which is a non-covalent interaction formed by the hydrogen bond between K7 and D69 [39][26][41].The other mechanism is oxidation-dependent, which is a covalent interaction formed by intermolecular disulphide bond between oxidation-sensitive Cys residues [29].Additionally, these two processes induce oligomerization independently.We identi ed TRIM44 promotes the oligomerization of SQSTM1 by enhancing these two interactions.As demonstrated in Fig. 4C, TRIM44 promotes the phosphorylation of two oxidation-de cient SQSTM1 mutations, indicating that TRIM44 increases the sensitivity of SQSTM1mediated autophagy, especially under oxidative stresses.
The phosphorylation of SQSTM1 at S349 is pivotal for KEAP1 binding.However, the kinase(s) responsible for this site have been unclear.The rst candidate kinase for this site is mTORC1.Inhibition of mTORC1 using rapamycin in mouse embryonic broblasts suppressed the phosphorylation of SQSTM1 at S349, as well as the expression levels of NFE2L2 targets [32].Another potential kinase is ULK1, which is recently reported [42], although knockout of Ulk1 and/or Ulk2 had no effect on this phosphorylation following oxidative stress [32].In our study, we did not observe any effect on the phosphorylation mediated by TRIM44 following inhibition of mTORC1 or ULK1 under basal and oxidative conditions (Supplementary Fig. S2D and F).Our ndings reveal that a novel kinase PKA is responsible for the increased phosphorylation of SQSTM1 at S349 triggered by TRIM44.
In this study, we demonstrate that TRIM44 promotes SQSTM1 oligomerization via its deubiquitinating activity.The oligomerization primed by TRIM44 is not only PB1 domain-dependent but also oxidationdependent.Furthermore, the oligomerization of SQSTM1 is essential for its phosphorylation at S349.PKA is responsible for the increased phosphorylation of SQSTM1 at S349 triggered by TRIM44, leading to more KEAP1 sequestration and then its degradation and subsequent activation of NFE2L2 under oxidative stress.This implies that TRIM44 might increase the sensitivity of SQSTM1-mediated autophagy under basal and oxidative conditions in cancer, ageing, ageing-associated diseases, and neurodegenerative diseases.
Importantly, this study also emphasized the role of TRIM44 in the regulation of autophagy in response to oxidative stress induced by As [III].Although the clinical successes of As[III] in treating hematological cancers have not been translated to solid cancers due to the quick removal of As[III] by the body's immune system, immense progress has been made in delivering As[III] compounds speci cally to cancer cells.Along with other arsenic compounds, As[III] has successfully been encapsulated in liposomes, polymersomes, and other nanoparticles.In addition, ligands speci c to the receptors overexpressed on cancer cells have been conjugated to these nanoparticles.Therefore, it is promising that delivering As[III] using nanotechnology would be applied as an anti-cancer drug option in treating a variety of solid cancers with improved e cacy and much less toxicity and it is essential to study the mechanisms by which cancer cells avoid apoptosis induced by As [III].Autophagy, functioning synergistically with the KEAP1-NFE2L2 system, establishes a robust defense against metabolic and oxidative stresses.These two pathways are intricately connected, as demonstrated by the phosphorylation of the ubiquitin-binding autophagy receptor protein SQSTM1 with the SQSTM1-KEAP1-NFE2L2 pathway [36].Our results showed that TRIM44 emerges as a crucial mediator in this interplay and promotes autophagy in response to As[III] induced oxidative stress which results in decreased cytotoxicity.

Cell lines
HeLa, HEK293T, U266 cell lines were obtained from ATCC (Rockland, MD).We veri ed these cell lines via STR pro ling (short tandem repeat analysis of DNA).Cell lines that contained knockout or overexpression constructs were not passaged beyond ~ 8 generations (1-1.5 months).

Plasmids
To construct SQSTM1 truncates, a series of deletions of SQSTM1 were ampli ed by PCR using appropriate primers and cloned into the HA empty vector.GFP-TRIM44, and its deletion mutant expression plasmids have been described previously [24].

Apoptosis assay
MM cells were irradiated with 5 µM As[III] and harvested after 24 h.The cells were stained with PE-Annexin V and 7-AAD (BD Bioscience, 559763) and examined with an LSR-II ow cytometer.The percentage of apoptosis was analyzed using FACS Diva software (BD Bioscience).

Confocal microscopy
The cells were xed with 4% paraformaldehyde (Thermo Fischer scienti c, AAJ61899AK) and permeabilized with 0.2% triton-X 100 (Sigma-Aldrich, 93443).Following blocking with Animal-Free Blocker (Vector laboratories, SP-5030-250) for 1 h, followed by washing with 1× phosphate buffer saline (PBS) (Corning, 46-013-CM; pH 7.4).The cells were then incubated with indicated antibodies overnight at 4°C, washed 3 times with PBS and incubated with uorochrome-conjugated secondary antibodies for 1 h at room temperature, washed 3 times with PBS and incubated with DRAQ5 (Cell Signaling Technology, 4084) for 30 min.Slides were analyzed by Confocal microscopy (Leica TCS SP5).To quantify indicated protein levels, four or ve random images were taken from each slide, and were quanti ed with Image J software (National Institutes of Health).

Immunoprecipitation and immunoblotting
Cells were lysed in NP-40-containing lysis buffer (BP-119, Boston Bioproducts, Ashland, MA, USA) containing protease inhibitors mixture (11697498001, Complete; Roche Diagnostics, Mannheim, Germany) and phosphatases inhibitors and incubated with indicated antibodies together with the protein A/G plus-agarose immunoprecipitation reagent (Santa Cruz Biotechnology, sc-2003) at 4°C overnight.Immunoprecipitates were eluted with Laemmli sample buffer (Bio-Rad) and analyzed using the indicated antibodies.
CHX chase assay MM cells were seeded into 6-well plates at a density of 3 × 10 5 cells/well and incubated overnight at 37°C in a CO 2 incubator.Cells were treated with 50 µg/ml of CHX dissolved in absolute ethanol, and harvested in ice cold phosphate buffered saline (PBS, pH 7.4) at varying chase points by centrifugation at 2500 × g for 10 min at 4°C. Cell pellets were lysed in a lysis buffer.Samples were heated at 95°C for 10 min.

Cell lysate fraction
Cells were lysed in a NP-40-containing lysis supplemented with protease inhibitors mixture and centrifuged at 15,000 x g into supernatant (Non-aggregates) and pellet (Aggregates) fractions.Both fractions were boiled in buffer containing 1% SDS and analyzed by western blot.

RNA extraction and real-time PCR
Total RNA was isolated using the Direct-zol™ RNA MicroPrep kit (Zymo Research, R2060), and cDNA was synthesized using random hexamers and RevertAid RT kit (Thermo Scienti c, K1691).Gene expression was determined using an ABI 7900 system (Applied Biosystems) with SYBR Green MasterMix Plus (Thermo Scienti c, K0221) and normalized to ACTB expression.Relative expression was calculated as 2 − (Ct Target − Ct Control) .

Nuclear fraction extraction
Nuclear fractions were extracted by kit (Active Motif, 40010).

Statistical analysis
Data were analyzed by using the unpaired two-tailed Student's t test, and P < 0.05 was considered statistically signi cant.The results are expressed as the mean ± SD from at least 3-5 independent experiments.(E) 293T cells were transfected with HA-SQSTM1 constructs and expressed to MG132 (5 μM) for a duration of 6 h.The treated cells were subsequently cross-linked with DSP (0.4 mg/ml, 4°C, 2 h) and prepared under either reducing conditions (with β-ME) or nonreducing condition (without β-ME).Cell lysates were subjected to immunoblotting for the indicated antibodies.Immunoprecipitation was executed using an anti-HA antibody and immunoblotting for SQSTM1 p-S349 and HA.